In the field of vaccination, first generation vaccines comprised only the antigen against which an immune response was desired. However, because the presence of an antigen alone is in most cases only weakly efficient, a second generation of vaccines was developed, wherein the vaccinating composition includes one or more adjuvants as immunomodulators to enhance this immune response.
Several different techniques have been reported for providing the adjuvant at the vaccination site, depending on the context of the immunisation.
In the context of antigen-based vaccines (as opposed to cell-based vaccines), a widely applicable technique used for providing the necessary adjuvant is simply to combine the antigen with the adjuvant in the vaccinating composition. The resulting composition is administered directly to the subject, thereby supplying the antigen and adjuvant in a simultaneous and co-localised manner.
This simple approach cannot however be used in all vaccination settings. For example, in most cancers, useful antigens are often not known. This is the case for most of the common cancers in human such as lung, colon, stomach, lymphoma, brain. Therefore, new immunisation strategies are needed such as a cell-based approach. Immunisation strategy involving cell-based vaccines, where the antigen(s) to which an immune response is required is (are) produced by whole cells implanted in a subject, necessitates the use of more elaborate techniques to ensure the efficient delivery of adjuvant.
One solution in this type of context is to directly inject the immunomodulator at the vaccination site. The immunomodulator may either be “naked”, or alternatively may be administered in a slow release formulation using pegylated, liposomal microspheres (International patent application WO 98/33520, filed by Bystryn). This strategy is however limited by technical and biochemical difficulties, as well as some degree of systemic release inducing potential toxicities.
An alternative approach which has been proposed to circumvent the problems arising from the direct injection technique is the use of “by-stander cells” to locally produce the immunomodulators. According to this approach, cells producing the adjuvant are implanted in proximity to the source of the antigen, thereby providing an efficient local release of adjuvant at the vaccine site. The efficacy of this approach has been demonstrated in mice where the vaccine is a mix of GM-CSF secreting fibroblasts and irradiated tumour cells (Aruga et al, 1997, Cancer Research, 57, 3230-3237).
However, even this approach is not totally without drawbacks. Indeed, for human immunization, it is known that multiple immunizations are required. Since syngeneic by-stander cells are not easily available, allogenic cells are used in the vast majority of cases. Consequently, after the first injection, the “by-stander cells” are recognised by the immune system of the host (allorecognition) and are rejected, thereby preventing further production of immunomodulator. In addition, the allorecognition of the “by-stander cells” jeopardizes the desired immune response against the antigenic substance of the vaccine.
In order to avoid this allorecognition, Borrello et al (Human Gene Therapy, 1999) has described a strategy in which the GM-CSF supplying cell is a cell line, K-562, which fails to express HLA class I or II antigens, potentially decreasing the magnitude of the alloresponses generated on repeated immunisations. The K-562 cells, engineered to secrete GM-CSF do not express MHC molecules on their surface. They are HLA negative. These cells are however human cancer cells and are highly sensitive to potent rejection mechanisms occurring without the involvement of HLA class I or II proteins. These defence strategies are less specific but very rapid and potent for cellular destruction. At least two subtypes of lymphocytes known as γε T cells or natural killer (NK) lymphocytes are known to attack and destroy foreign cells by mechanism independent of HLA class I or II. Regarding K-562 they are known to be very sensitive to NK cells and also to γδ T cells (J. Immunotherapy 2000 23:536-548 Schilbach K. et al) and are therefore used as a positive control for NK cell activity.
It is therefore likely that K-562 by-stander cells injected at the vaccine site will be destroyed efficiently and quickly by non-MHC dependent cytotoxic mechanisms. This may very well significantly decrease the release of the immunomodulator.
Beside being very sensitive to rapid destruction by NK cells, K-562 cells can express MHC class I upon Interferon γ exposure. It is possible that such cytokine could be present or released at the vaccination site during the first or after repeated immunizations. Such MHC class I upregulation will also lead to rapid cell destruction via classical cellular immunity.
For these reasons, use of cells such as K-562 in vaccination is associated with numerous drawbacks.
Another solution, widely used in the context of cell-based vaccines, for example in cancer therapy, is to couple the production of antigen and the release of immunomodulator, by engineering the cell which is the source of antigen, to also supply the immunomodulator. For example, in cancer vaccines, the source of antigen is usually a whole tumour cell. This cell can be engineered, for example by transfection, to simultaneously produce the necessary adjuvant. Potent, specific and long lasting anti-tumour immune responses have been reported in the mouse model using this technique, relying on retroviral vectors as the gene transfer method for engineering tumour cells delivering GM-CSF.
In view of the favourable results obtained in the mouse model, the initial human trials used the same strategy (Simons J W. et al. 1997 Cancer Research, 43(11); Soiffer R. et al. 1998, Proc. Natl. Acad. Sci. USA, 95, 13141; Simons et al. 1999, Cancer Research, 59(20)). However, the technique proved to be very labour intensive and time consuming, because the patient's cells, harvested surgically need to be expanded in vitro, for retroviral infection, preventing a wide use of the method.
The use of other viral vector for infecting the tumour cells has therefore been proposed to circumvent these difficulties. Engineered viruses like adenovirus can infect tumour cells very efficiently and with much simpler procedures. Because adenovirus can infect non-dividing cells, the harvested tumour cells can be infected right away, preventing the long and tedious primary culture step required when using retroviral vectors.
The major problem of the new viruses tested is that in most cases, some viral proteins will be expressed from the tumour cells after infection. These viral proteins are strongly recognized by the immune system as foreign, infectious agents. Therefore the initial goal of mounting an immune response against weak tumour antigens is skewed or diverted towards a viral protein. This results in masking the anti-tumour immune response. It also primes the recipient against subsequent immunization that will further increase the destruction of the injected cells. These two mechanisms will very likely decrease the efficacy of the anti-tumour immunization scheme.
Thus, whilst the use of autologous engineered tumour cells as combined source of antigen and adjuvant a priori minimizes the risk of undesirable immune response, the step of viral infection itself gives rise to significant problems.
In order to limit the problems arising from viral infection of autologous cells, new strategies have been developed which do not require patients' cells. According to these techniques, the antigenic source is provided by cell-lines derived from other patients with similar type of cancer. The patient is then immunized with repeated injections of irradiated, GM-CSF secreting, allogeneic (from another human being) tumour cells.
This strategy is based on the assumption that the cell-line used for the immunization shares some relevant antigens with the patient's own tumour and that these common antigens will allow the development of an immune response that will recognize the patient tumour cells and destroy them. A phase I clinical trial using such strategy has been published (Jaffee E. et al. 2001 Journal of Clinical Oncology, 19(1)).
However, the percentage of patients showing an immune response is much lower than in previous report using autologous cells secreting the same immunomodulator (GM-CSF) (Soiffer et al.).
To date, no technique has been reported which provides both a constant source of immunomodulator and an efficient immune response, whilst being substantially free of undesirable interactions with the natural or adaptative immune system.